Well-defined and novel ubiquitin derivatives would be invaluable tools for basic research and drug discovery efforts, but these are commercially unavailable. We propose in this Phase I SBIR grant to develop a comprehensive panel of ubiquitin variants, making all combinations of lysine mutations and N-terminal modifications, as well as a collection of N-terminal epitope and hapten tags. Each ubiquitin will be tested for competence in E1 activation and E2 conjugation assays. The panel of unmodified and modified ubiquitin proteins will be used to produce unanchored polyubiquitin chains with unique lysine linkages, using various ubiquitin conjugation enzymes systems. Methods to synthesize, scale-up, purify to homogeneity and characterize alternate chain linkages in vitro will be developed. These polyubiquitin chain substrates will be extensively characterized in relevant and novel biochemical assays. They will finally be tested for hydrolysis against a battery of deubiquitinating enzymes, including Isopeptidase T, UCH-L1, UCH-L3, BAP1, and USP14, to characterize these hydrolytic specificities of these enzymes. Once fully characterized, these novel Ubiquitin proteins and polyubiquitin chains will be commercialized in Phase II and made available to the entire research community as both free standing reagents, and as part of a broader assay platform for designing inhibitors of any deubiquitination enzyme. This work will also result in the ability to produce, at commercial scale, ubiquitin chains using comprehensive linkages and modification types. This defined product suite creates invaluable options for new research tools and assays, which will facilitate and accelerate discovery and target research efforts. Such a panel of proteins would revolutionize the speed and ease of ubiquitin-related research endeavors, facilitate the further understanding of fundamental questions about misdirected or abnormal ubiquitination processes, and aid in the discovery of new therapeutics, especially as regards to deubiquitination and the proteasome. [unreadable] [unreadable]